Purification of antibodies

ABSTRACT

Recovering the entire spectrum of all heterogeneous antibodies against a specific antigen or ligand by a process of adding an antigen to an immune serum forming a precipitate which is dissociated by a combination of addition of excess hapten and a non-specific dissociating compound and separating the dissociated heterogeneous antibodies from the antigen, hapten and dissociating compounds added.

United States Patent 3,697,645

Meier et al. Oct. 10,1972

[54] PURIFICATION or ANTIBODIES Inventors: Eugene P. Meier, Edgewood;David E. Lenz, Bel Air; Ludwig A. Sternberger, Lutherville, all of Md.

The United States of America as represented by the Secretary of the ArmyFiled: April 28, 1971 Appl. No.: 138,354

Assignee:

Primary Examiner-Richard L. Huff AttorneyHarry M. Saragovitz, Edward J.Kelly, Herbert Berl and Jacob Ziegler [5 7] ABSTRACT Recovering theentire spectrum of all heterogeneous antibodies against a specificantigen or ligand by a process of adding an antigen to an immune serumforming a precipitate which is dissociated by a combination of additionof excess hapten and a nonspecific dissociating compound and separatingthe dissociated heterogeneous antibodies from the antigen, hapten anddissociating compounds added.

1 1 Claims, No Drawings PURIFICATION OF ANTIBODIES DEDICATORY CLAUSE Theinvention described herein may be manufactured, used, and licensed by orfor the Government for governmental purposes without the payment to usof any royalty thereon.

This invention describes methods of obtaining a 100 percent recovery ofa specific antibody population from antiserum for the prevention of theadverse effects of various antigens or ligands including poisons inmammals. The antibody population or fraction contains all the propertieswhich can be associated with antibodies to an antigen or ligand, forexample, nerve agents.

The object of this invention is to provide a purified antibody fractioncontaining the entire spectrum of all the heterogeneous antibodiesagainst a specific antigen or ligand.

It is a further object ofthis invention of obtaining the total purifiedantibody population containing both the poorer binding and the strongerbinding portions in the same proportions in which they have been presentin the serum.

L. A. Sternberger et al, J. Immuno., 65, 65, 1950, utilized the methodof antibody separation employing alkali for dissociation of the immuneprecipitate and giving rise to a specific antibody population whichcontains only a portion of the total specific antibodies.

Antibodies have been dissociated from immunoabsorbents by interferringwith either the coulombic or hydrogen bonding or hydrophobic bondingbetween antigen and antibody by separately using urea increased saltconcentration (salt gradient), gradient), alkaline pH, acid pH, or anexcess of antigen. S. F. Schlossman et al., J. Exper. Med., 116, 535,1962; De St Groth, Ann. N. Y. Acad. Sci., 103, 609, 1963; M. E. Kaplinet al., J. Exper. Med., 123, 106, 1966, have shown that the separationof specific antibodies from antigen utilizing immunoabsorbents resultsin a recovery of a portion of the specific antibody popula tion, that isan antibody population which is not heterogeneous but rather possessesthe poorer binding properties with the antigen while leaving on theabsorbent an antibody population having the strongest bindingproperties.

The major deficiency of the prior art is that not a single method canrecover the entire heterogeneous specific antibody population but thereis present an antigen-antibody entity which prevents the total recoveryof the desired antibodies.

As a result of the previous methods not achieving total recovery of allthe antibody population to a specific antigen or ligand in theantiserum, we launched upon an investigation for total recovery of theantibody population with the entire spectrum of antibody being availableto the investigator and final utilization by the clinician to counteractthe deleterious effect of various chemical compounds.

Our inventive procedure permits for the first time recovering the totalantibody fraction for a specific antigen or ligand by dissociatinghydrophobic and coulombic bonds between antigen and antibody andutilizing a non-specific antibody such as antihemocyanin to removeextraneous antigen. The procedure is directed to the final rupturing ofthe bonding effect between antigen-antibody without any subsequentreassociation of the antigen with the antibody and then antibodyseparation thus giving rise to the whole antibody fraction to a specificmoiety system, for example, aromatic acids, sugars, organophosphates,small peptides or neutral antigenic determinants. The process ifapplicable to various moieties containing not more than one antigenicvalence.

The molecular moieties which contain not more than one antigenic valenceare, for example, acids of arsanilic or benzoic containing the aromaticnucleus, saccharides of glucosamine or galactose, peptides ofglycyclglycine, glycyltryrosine and other peptides containing up to 10aminoacid residues, nucleotides containing up to seven nucleosideresidues, neutral antigenic determinants of dinitrophenol,trinitrophenol,

dinitrophenylysine, or dinitrophenylcaproate; organophosphates(G-Agents) Z-methylcyclohexyl methylphosphonofluoridate, isopropylmethylphosphonofiuoridate, pinacolyl methylphosphonofluoridate,cyclohexyl methylphosphonofluoridate, and other organophosphate agents0,0-diethylpara-nitrophenylthiophosphate (parathion), insecticide, andpnitrophenyl diethylphosphate (paraoxon), insecticide, nerve gas.

A general method for the preparation of antibodies to paraoxon,p-nitrophenyl diethylphosphate. An antigen prepared by diazotizingp-aminophenyldiethylphosphate to a carrier of hemocyanin is injectedinto a living animal; the carrier is a protein with a molecular weightin excess of 500,000, and an animal can be any animal used forimmunization, i.e., rabbit. The animal serum is pooled and containsantibodies to p-aminophenyldiethylphosphate (hereinafter called anti-L)and several species of antibodies to hemocyanin (hereinafter calledanti-M). Said antigen is added to the serum forming an immuneprecipitate which comprises the antigen, anti-L, and anti-M. The immuneprecipitate is resuspended on dissolution between 0 to 56 C. in thepresence of 0.001 M to 10.0 M, an excess of p-aminophenyldiethylphosphate (hapten, hereinafter called L) and then adding acompound which causes dissolution of the precipitate forming a solutionof free anti-L, free antigen, free L and fee anti- M; the time fordissociation can be instantaneous or may be pro longed for hours ordays. The dissolution step may be carried out in one of several manners,for example acidic pH 1 to 4, alkaline pH 11 to 13, or salt addition ofurea at 5M to 7M; an acid can be l-ICl and alkaline substance of alkalimetal hydroxide of sodium or potassium; when employing salt additiontechnique dialysis against water or any buffer physiologic to theantibody is to be utilized. The solution containing the dissociatedimmune precipitate is neutralized to pH 7 to 7.6 thereby the free anti-Mcombines with the free antigen removing the antigen from solution byprecipitation forming soluble complexes of anti-L with a part of thelarge excess of free L. The solution containing the soluble complexes isdialyzed against water or any buffer physiological to the anti-L, forexample, 0.15 M NaCl and 0.01 M sodium phosphate, pH 7.4, described bySinger, S. J., et al., J. Am. Chem. Soc., 82, 565 (1960), (1960), thusseparating the purified anti-L from L, and anti-L can be stored at 1 C.,frozen or lyocold, frozen or lyophilized; the supernatant afterneutralization to pH 7 to 7.6, cold storage and centrifugation comprisesanti-L and hapten; the dextran possesses water regain of 10 i 1.0 g/gdry gel), bed volume of 100,000, fractionation range of 1,000-l00,000,particle size of 40 to 120 microns, for example, Sephadex, Superfine G-l00.

The antigen used is reactive with two species of antibodies in theimmune serum; only one species is intended for purification. The secondspecies which is not intended to be purified reprecipitates the antigenupon completion of the procedure thus leaving pure antibody to the firstspecies. 5

In addition to the procedures described in this specification, it isalso an objective of this invention to employ methods for immunizinganimals, preparing pooled serum, compounds containing the group ofpaminoaryl (p-aminophenyl) or diazoaryl and antigen described in theliterature of the prior art, supra, for antibody separation; the use ofother ligands than paraoxon and the mode of chemical conjugation forantigen preparation described by Pressman, D., and Sternberger, L. A.,J. ImmunoL, 66, 609, (1951); (1951); and Holtschmidt, US Pat. No.3,013,048, describing preparation for p-aminophenyl diethylphosphate bycatalytic reduction of the nitro group in p-nitrophenyldiethylphosphate, paraoxon.

The production of antibodies against paraoxon, diethyl-p-nitrophenylphosphate, in accordance with the procedure of Example 1.

EXAMPLE 1 a. The animals are imunized with an aqueous solutioncomprising the antigen of diazotized p-aminophenyldiethylphosphatecoupled into hemocyanin. Each animal, rabbit, is injected with 10 mg/kgon a weekly basis over a period of 3 to 4 weeks. Subsequently eachanimal is bled on a weekly basis. The red cells are removed by gravityprecipitation to prevent hemolysis and the collected serum pooled. Saidserum (19 ml) is combined with 27.14 ml of a solution, 10 grams ofdiazotized p-aminophenyl diethylphosphate coupled into hemocyanin perliter of 0.15N saline. The solution which results is placed in the cold(4C.) for 48 hours giving rise to a precipitate which is subsequentlycollected. The latter precipitate (1 to 3 grams) is resuspended in aboutml of 0.1 M p-aminophenyldiethylphosphate-HCI forming a suspension whichis adjusted to pH 2.3 (BC!) with stirring at room temperature withcomplete dissolution of the precipitate forming a solution which isneutralized to approximately pH 7.4. An aqueous solution comprising 0.15N in sodium acetate and 0.3 N in ammonium acetate is added to thesolution at pH 7.4 in the ratio of l to 10 respectively and theresulting solution placed in he cold (4C.) for about 4 days after whichtime any insoluble material is removed by centrifugation and thesupernatant collected containing the antibodies to paraoxon. Thesupernatant is dialyzed against several changes of saline (0.15 N sodiumchloride) containing 0.015 N sodium acetate and 0.03 N ammonium acetate.The final dialyzed material inside the semipermeable mem brancecontaining all the antibodies in a liquid medium todiethyl-p-nitrophenyl phosphate, paraoxon. The liquid medium is storedat 1 C., quick frozen or lyophilized.

b. The method according to a, supra, was repeated with the exception ofsubstituting for the conjugate of diazotizedp-aminophenyldiethylphosphate coupled into hemocyanin an antigenselected from the group consisting of diazotized 0-(p-aminophenyl)-0'-pinacolyl methylphosphonate coupled into hemocyanin, diazotizedp-aminobenzoic acid coupled into hemocyanin, diazotized glucosaminecoupled into hemocyanin, diazotized glycylglycine coupled intohemocyanin, diazotized p-amino-m-nitrophenol coupled into hemocyanin,and diazotized 0,0'-diethyly-0"- p-aminophenylthiophosphate coupled intohemocyanin, substituting for the addition of 0.1 Mpaminophenyldiethylphosphate' HCl in order for resuspension thecorresponding compound selected from the group consisting0-(p-aminophenyl)-0- pinacolyl methylphosphonate HC], p-aminobenzoicacid HCl, glucosamine HCl, glycylglycine HCl, pamino-m-nitrophenl l-lCl,and 0,0-diethyl-0"-paminophenylthiophosphate HC] and recovering thefinal dialyzed material of antibodies against compounds selected fromthe group consisting of O-pinacolyl methylfluorophosphonate, benzoicacid, glucosamine, glycylglycine, dinitrophenol and0,0'-diethyl-0"-pnitrophenylthiophosphate. In the preparation of theantigen the carrier can be any protein having a molecular weight inexcess 500,000, for example hemocyanin, ferritin, copolymers of proteinssuch as polyimmunogloblin or polyalbumin, such polymers being producedwith tetraazotized benzidine, glutaraldehyde, bis (flurodinitrobenzene)sulfone or other bifunctional or multifunctional protein reagents.Preparation of the diazotized p-aminophenyldiethylphosphate intohemocyanin in Example 2.

EXAMPLE 2 a. Three moles of HCL and one mole ofpaminophenyldiethylphosphate were diazotized at 5 to 10 C. with asolution comprising 0.1 to 0.5 molar of sodium nitrite, to the starchiodine end point in the conventional procedure, producing the diazotizedpaminophenyldiethylphosphate. An excess of said diazotizedp-aminophenyldiethylphosphate was added to a carrier of hemocyanin inthe molar ratio of 400021 respectively forming a mixture which wasallowed to stand and react at room temperature for approximately onehour buffered to a pH of 7.2 using 12.5 percent, weight/volume, ofsodium carbonate producing the antigen of diazotizedp-aminophenyldiethylphosphatehemocyanin. The antigen was purified by gelfiltration on dextran at 2 to 4 C. removing the impurities with 0.028 Msodium cacodylate as the eluting medium and recovering the antigen inthe chant; the filter medium being any dextran having a fractionationrange of to 5000 molecular weight, and exclusion limit of 5,000molecular weight, and 2.5 i 0.2 grams of water per gram of dry gel waterregain.

b. The method in accordance with he procedure under (a) was repeatedwith the exception of substituting for the p-aminophenyldiethylphosphate a compound selected from the group consisting of0(paminophenyl)-0-pinacolyl methyl phosphate, paminobenzoic acid,glucosamine, glycylglycine, pamino-m-nitrophenyl, and0,0'-diethyl-0"-paminophenylthiophosphate and producing the antigen ofdiazotized compounds coupled into hemocyanin enumerated in Example 1(b).

Table 1 below describes the percent fluorescence of antibody populationcompared with varying concentrations of paraoxon utilizing the followingbuffered solutions of antibody and paraoxon. The antibody sample is 1.07X M of lyophilized antibody in sodium phosphate buffer, pH 6.8, ionicstrength 0.1 in sodium chloride. The antibody has an average molecularweight of 148,000 and was prepared in accordance with Example 1. The1.07 X 10"" M nerve agent sample is prepared by dissolving 0.029 gramsof diethyl pnitrophenyl phosphate, paraoxon, in a final volume of 100 mlof sodium phosphate buffer, pH 6.8, containing 6 ml of isopropylalcohol.

The fluorescence study was made on an Aminco- BowmanSpectrophotofluorometer at an excitation wavelength of 280Spectrophotofluorometer ,u. and an emission wave length of 335 [.Lemploying an 0.2 ml sample.

"LD, is 350 mg/kg in mammals i.e. man. Ratio of Paraoxon to Antibody (a)(80.3X10'9)/(5.35X10l0) 150 (b) (107.0X10'9)/(5.35 10'10)= 200 averageRatio= 175 The data from Table l was treated in accordance with themethod of Stockell, A., J. Bio. Chem, 234, 1286 (1959), which gave anestimation of the minimum number, 50, of sites involved in binding andan estimate of the average association constant per site of 10'5 M 1.

The data demonstrate that the interaction of a ligand, i.e., paraoxon,plus antibody takes place at the average mole ratio of to 1respectively. This ratio 175:1 is about 87 times greater than thosereported for ligand and antibody interaction, i.e., 2 to l,respectively, reported by Velick, S. F. et al., Proc. Nat. Acad. Sci.,US. 46, 1470 (1960). The mole ratio of 2 to 1 obtained using antibodiesisolated by prior art methods, for example, Velick, supra, isinsufficient to counteract the ligand. The publication data as comparedwith the data from Table 1, above, indicate that the mole proportion ofantibody combining with l/87 of the proportion of ligand would not giveprotection against one LD (350 mg/kg in mammals, i.e., man). Theheterogeneous antibody population of this invention now makes availablea greater proportion of antibody to interact with greater quantities ofligand, i.e., 87 times the amount previously reported. The fluorescencedata indicate the antibody fraction contains the entire spectrum ofantibody population, that is, the combination of the poorer and strongerbinding properties present in the original antiserum. The recoverableproportion of the antibody population by the prior art procedures makesavailable only the population with the poorer binding properties thusless effective in combining with ligand. The clinician has now availablea more effective antibody population against multiple LD levels ofpoisoner by harnessing the total population against a specific nerveagent.

We claim:

1. A method for isolating the total heterogeneous antibody populationagainst antigens, the steps comprising adding an antigen to an immuneserum comprising antibody to said antigen and thereby forming an immuneprecipitate in an aqueous medium, dissociating said precipitate byadding in combination an excess of a hapten or the antigen and acompound selected from the group consisting of acid, alkali and salt,neutralizing the aqueous medium thereby forming an insolubleantigen-antibody precipitate and a soluble complex of antibody with thehapten or the antigen, removing the insoluble antibody-antigenprecipitate, separating the antibody from the soluble complex therebyrecovering a heterogeneous antibody population against moieties oforganophosphates, aromatic acids, sugars, small peptides or neutralantigenic determinants.

2. The method according to claim 1, wherein the acid is HCl and theliquid medium containing the dissociated precipitate is at pH 1 to 4.

3. The method according to claim 1, wherein the alkali is an alkalinemetal hydroxide and the liquid medium containing the dissociatedprecipitate is at pH 11 to 13.

4. The method according to claim 1, wherein the salt is urea and theliquid medium is 5 molar to 7 molar in said salt.

5. The method according to claim 1, wherein the antigen added to theimmune serum is selected from the group consisting of diazotizedp-aminophenyl diethylphosphate coupled into hemocyanin, diazotized0-(p-aminophenyl)-0-pinacolyl methylphosphonate coupled into hemocyanin,diazotized p-aminobenzoic acid coupled into hemocyanin, diazotizedglucosamine coupled into hemocyanin, diazotized glycylglycine coupledinto hemocyanin, diazotized p-amino-mnitrophenol coupled intohemocyanin, and diazotized 0,0'-diethyl-0"-p-aminophenylthiophosphatecoupled into hemacyanin, the hapten is selected from the groupconsisting of p-aminophenyl diethylphosphate HCl,(p-aminoplienyl)-O-pinacolyl methylphosphonate HCl, p-aminobenzoic acidHCl, glucosamine HCl, glycylglycine HCl, p-amino-m-nitrophenyl HCl, and0,0'-diethyl-0"p-aminophenylthiophosphate HCl, an the moieties areselected from the group consisting of diethyl-p-nitrophenyl phosphate,o-pinacolyl methylfluorophosphate, benzoic acid, glucosamine,glycylglycine, dinitrophenol and0,0'-diethyl-O-pnitrophenylthiophosphate.

6. The method according to claim 5, wherein the step of separating thesoluble complex is gel filtration.

7. The method according to claim 5, wherein the step of separating thesoluble complex is dialysis.

8. The method according to claim 7, wherein the antigen is diazotizedp-aminophenyl diethylphosphate coupled into hemocyanin, the hapten isp-aminophenyl diethylphosphate l-lCl and the moiety of organophosphateis diethyl-p-nitrophenyl phosphate.

9. The method according to claim 7, wherein the antigen is diazotized0-( p-aminophenyl)-0-pinacolyl methylphosphate coupled into hemocyanin,the hapten is O-(p-aminophenyl)-0'-pinacolyl methylphosphate HCl and themoiety of organophosphate is O-pinacolyl methylfluorophosphate.

10. The method according to claim 7, wherein the antigen is diazotized0,0-diethyl-0"-p-aminophenylthiophosphate coupled into hemocyanin, thehapten is 0,0'-diethyl-O"-p-aminophenylthiophosphate HCI, and the moietyof organophosphate is 0,0'-diethyl-0"- p-nitrophcnylthiophosphate.

11. A total heterogeneous antibody population against a specificpoisoner produced by adding an antigen to an immune serum comprisingantibody to said antigen and forming an immune precipitate in an aqueousmedium, dissociating said precipitate with excess hapten, adding to theaqueous medium comprising the dissociated precipitate a compound ofacid, alkali or salt preventing reassociation of the dissociatedprecipitate and thereafter neutralizing the aqueous medium forming aninsoluble antigen and soluble complex of antibody with hapten,centrifuging the neutralized aqueous medium and removing the insolubleantigen with the medium consisting essentially of the soluble complex,separating the antibody from the hapten by dialyzing the soluble complexin a semipermeable membrane against a physiologic buffer to the antibodyand removing the aqueous medium consisting essentially of theheterogeneous antibody population from the interior of the membrane soobtained from dialysis.

2. The method according to claim 1, wherein the acid is HCl and theliquid medium containing the dissociated precipitate is at pH 1 to
 4. 3.The method according to claim 1, wherein the alkali is an alkaline metalhydroxide and the liquid medium containing the dissociated precipitateis at pH 11 to
 13. 4. The method according to claim 1, wherein the saltis urea and the liquid medium is 5 molar to 7 molar in said salt.
 5. Themethod according to claim 1, wherein the antigen added to the immuneserum is selected from the group consisting of diazotized p-aminophenyldiethylphosphate coupled into hemocyanin, diazotized0-(p-aminophenyl)-0''-pinacolyl methylphosphonate coupled intohemocyanin, diazotized p-aminobenzoic acid coupled into hemocyanin,diazotized glucosamine coupled into hemocyanin, diazotized glycylglycinecoupled into hemocyanin, diazotized p-amino-m-nitrophenol coupled intohemocyanin, and diazotized0,0''-diethyl-0''''-p-aminophenylthiophosphate coupled into hemacyanin,the hapten is selected from the group consisting of p-aminophenyldiethylphosphate . HCl, 0-(p-aminophenyl)-0-pinacolyl methylphosphonate. HCl, p-aminobenzoic acid . HCl, glucosamine . HCl, glycylglycine .HCl, p-amino-m-nitrophenyl . HCl, and0,0''-diethyl-0''''p-aminophenylthiophosphate . HCl, an the moieties areselected from the group consisting of diethyl-p-nitrophenyl phosphate,o-pinacolyl methylfluorophosphate, benzoic acid, glucosamine,glycylglycine, dinitrophenol and0,0''-diethyl-0''''-p-nitrophenylthiophosphate.
 6. The method accordingto claim 5, wherein the step of separating the soluble complex is gelfiltration.
 7. The method according to claim 5, wherein the step ofseparating the soluble complex is dialysis.
 8. The method according toclaim 7, wherein the antigen is diazotized p-aminophenyldiethylphosphate coupled into hemocyanin, the hapten is p-aminophenyldiethylphosphate . HCl and the moiety of organophosphate isdiethyl-p-nitrophenyl phosphate.
 9. The method according to claim 7,wherein the antigen is diazotized 0-(p-aminophenyl)-0''-pinacolylmethylphosphate coupled into hemocyanin, the hapten is0-(p-aminophenyl)-0''-pinacolyl methylphosphate . HCl and the moiety oforganophosphate is 0-pinacolyl methylfluorophosphate.
 10. The methodaccording to claim 7, wherein the antigen is diazotized0,0''-diethyl-0''''-p-aminophenylthiophosphate coupled into hemocyanin,the hapten is 0,0''-diethyl-0''''-p-aminophenylthiophosphate . HCl, andthe moiety of organophosphate is0,0''-diethyl-0''''-p-nitrophenylthiophosphate.
 11. A totalheterogeneous antibody population against a specific poisoner producedby adding an antigen to an immune serum comprising antibody to saidantigen and forming an immune precipitate in an aqueous medium,dissociating said precipitate with excess hapten, adding to the aqueousmedium comprising the dissociated precipitate a compound of acid, alkalior saLt preventing reassociation of the dissociated precipitate andthereafter neutralizing the aqueous medium forming an insoluble antigenand soluble complex of antibody with hapten, centrifuging theneutralized aqueous medium and removing the insoluble antigen with themedium consisting essentially of the soluble complex, separating theantibody from the hapten by dialyzing the soluble complex in asemipermeable membrane against a physiologic buffer to the antibody andremoving the aqueous medium consisting essentially of the heterogeneousantibody population from the interior of the membrane so obtained fromdialysis.